Cytometry By Time-of-flight Research Articles

Lianhua Qingke Tablet in Severe Pneumonia: Clinical Efficacy and Immunoregulatory Mechanisms.

Lianhua Qingke (LHQK), a traditional Chinese medicine, has shown efficacy in treating acute and chronic bronchitis and bronchiolitis. However, the specific mechanism underlying the therapeutic effects of LHQK on severe pneumonia is not clear. Severe pneumonia remains a critical health challenge, particularly in cases progressing to sepsis and septic shock, where host immune responses become dysregulated or dysfunctional. This study aims to evaluate the immunomodulatory effects of LHQK in severe pneumonia. This research examined LHQK's therapeutic and immunomodulatory mechanisms in patients with severe pneumonia and a lipopolysaccharide (LPS)-induced mouse model of severe pneumonia. Patients with severe pneumonia were randomized into three groups: basal treatment, LHQK-Low dose (12 tablets/day), and LHQK-High dose (24 tablets/day). BALB/c mice were categorized into four groups: control, model, LHQK-Low dose (3.7 mg/kg), and LHQK-High dose (7.4 mg/kg). Clinical efficacy was evaluated by assessing parameters including the value and rate of change in APACHE II score, improvement in chest X-ray or CT, partial pressure of oxygen (PO2), oxygen saturation in arterial blood (SaO2), oxygenation index (OI), and the length of hospitalization after 7 days of treatment. The viscosity of sputum was measured by viscosimeter. Moreover, lung histopathology, airway barrier integrity, and immune cells in BALF, were assessed using hematoxylin and eosin staining, immunostaining, and Wright-Giemsa staining. Cytokine levels were measured using Luminex assay and Olink, while pulmonary immune cell patterns were analyzed using multiplex fluorescence and Cytometry by Time-Of-Flight (CyTOF). In comparison to the basal treatment group of patients, LHQK treatment exhibited a reduction in the severity of severe pneumonia and inflammatory status, as evidenced by observations on Chest X-ray or CT scans. Additionally, LHQK treatment led to an elevation in OI, PO2, and SaO2 levels, and notably, a decreased duration of hospitalization. Further analysis revealed that LHQK enhanced the integrity of the airway epithelial barrier, reduced the viscosity of sputum, and significantly decreased inflammatory cells infiltration. The application of Luminex and Olink assay further confirmed the inhibitory impact of LHQK on the cytokine storm in mice. Moreover, multiplex fluorescence and CyTOF analysis demonstrated that LHQK effectively suppressed the activation of monocyte derived macrophages, neutrophils, and Treg cells, while preserved the levels of alveolar macrophages, B cells, and CD4+ and CD8+ T lymphocytes, therefore restoring immune homeostasis within the lung of severe pneumonia. These findings significantly substantiate the potential clinical application of LHQK in severe pneumonia treatment. LHQK demonstrates therapeutic efficacy in severe pneumonia by maintaining structural integrity, suppressing cytokine storms, enhancing intrinsic immunity, reversing T cell exhaustion, and correcting lung immune disorders. These findings significantly substantiate LHQK's potential clinical application in severe pneumonia treatment.

Oncogenic IDH1mut drives robust loss of histone acetylation and increases chromatin heterogeneity

Malignant gliomas are heterogeneous tumors, mostly incurable, arising in the central nervous system (CNS) driven by genetic, epigenetic, and metabolic aberrations. Mutations in isocitrate dehydrogenase (IDH1/2mut) enzymes are predominantly found in low-grade gliomas and secondary high-grade gliomas, with IDH1 mutations being more prevalent. Mutant-IDH1/2 confers a gain-of-function activity that favors the conversion of a-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), resulting in an aberrant hypermethylation phenotype. Yet, the complete depiction of the epigenetic alterations in IDHmut cells has not been thoroughly explored. Here, we applied an unbiased approach, leveraging epigenetic-focused cytometry by time-of-flight (CyTOF) analysis, to systematically profile the effect of mutant-IDH1 expression on a broad panel of histone modifications at single-cell resolution. This analysis revealed extensive remodeling of chromatin patterns by mutant-IDH1, with the most prominent being deregulation of histone acetylation marks. The loss of histone acetylation occurs rapidly following mutant-IDH1 induction and affects acetylation patterns over enhancers and intergenic regions. Notably, the changes in acetylation are not predominantly driven by 2-HG, can be rescued by pharmacological inhibition of mutant-IDH1, and reversed by acetate supplementations. Furthermore, cells expressing mutant-IDH1 show higher epigenetic and transcriptional heterogeneity and upregulation of oncogenes such as KRAS and MYC, highlighting its tumorigenic potential. Our study underscores the tight interaction between chromatin and metabolism dysregulation in glioma and highlights epigenetic and oncogenic pathways affected by mutant-IDH1-driven metabolic rewiring.

Targeting Deltex E3 Ubiquitin Ligase 2 Inhibits Tumor-associated Neutrophils and Sensitizes Hepatocellular Carcinoma Cells to Immunotherapy.

Several E3 ligases have been found to affect the immune microenvironment of hepatocellular carcinoma (HCC) and lead to the resistance of immunotherapy. In this study, genes of E3 ligases are screened based on The Cancer Genome Atlas (TCGA) dataset. Through cytometry by time of flight (CyTOF), flow cytometry, and further experiments, Deltex E3 ubiquitin ligase 2 (DTX2) in HCC cells is identified to promote the infiltration and polarization of tumor-associated neutrophils (TANs) with a protumor phenotype, thus attenuating the infiltration and cytotoxicity of CD8+ T cells partially through C-X-C motif chemokine 2 (CXCL2) and C-X-C motif chemokine 6 (CXCL6). Mechanistically, DTX2 can interact with histone H2B and promote its monoubiquitination at lysine120 (H2BK120ub1), thereby increasing CXCL2 and CXCL6 transcription through histone epigenetic regulation. Different tumor models in vivo demonstrated that DTX2 inhibitor treatment inhibited tumor growth and sensitized HCC cells to the therapeutic effects of programmed cell death protein 1 (PD-1) antibody. In summary, this study identifies DTX2 as a potential target for HCC immunotherapy.

Combined Bulk and Single-Cell Transcriptomic Analysis to Reveal the Potential Influences of Intestinal Inflammatory Disease on Multiple Sclerosis.

Multiple sclerosis (MS) and inflammatory bowel disease (IBD) are both autoimmune disorders caused by dysregulated immune responses. Still, there is a growing awareness of the comorbidity between MS and IBD. However, the shared pathophysiological mechanisms between these two diseases are still lacking. RNA sequencing datasets (GSE126124, GSE9686, GSE36807, GSE21942) were analyzed to identify the shared differential expressed genes (DEGs) for IBD and experimental allergic encephalomyelitis (EAE). Other datasets (GSE17048, GSE75214, and GSE16879) were downloaded for further verification and analysis. Shared pathways and regulatory networks were explored based on these DEGs. The single-cell transcriptome of central nervous system (CNS) immune cells sequenced from EAE brains and the public datasets of IBD (PRJCA003980) were analyzed for the immune characteristics of the shared DEGs. Mass cytometry by time-of-flight (CyTOF) of peripheral blood mononuclear cells (PBMCs) was performed for the systematic immune response in the EAE model. Machine learning algorithms were also used to identify the diagnostic biomarkers of MS. We identified 74 common DEGs from the selected RNA sequencing datasets, and single-cell RNA data of the intestinal tissues of IBD patients showed that 56 of 74 DEGs were highly enriched in IL1B+ macrophages. These 56 DEGs, defined as inflammation-related DEGs (IRGs), were also highly expressed in pro-inflammatory macrophages of EAE mice and MS patients. The abundance of systematic CD14+ monocytes was validated by CyTOF data. These IRGs were highly enriched in immune response, NOD-like receptor signaling pathway, IL-18 signaling pathway, and other related pathways. In addition, 'AddModuleScore_UCell' analysis further validated that these IRGs (such as IL1B, S100A8, and other inflammatory factors) are highly expressed mainly in pro-inflammatory macrophages, which play an essential role in pro-inflammatory activation in IBD and multiple sclerosis, such as IL-17 signaling pathway, NF-kappa B signaling pathway, and TNF signaling pathway. Finally, suppressors of cytokine signaling 3(SOCS3) and formyl peptide receptor 2(FPR2) were identified as potential biomarkers by machine learning. Two genes were highly expressed in pro-inflammatory macrophages of IBD and MS disease compared to control, and other datasets and experiments further revealed that SOCS3 and FPR2 were highly expressed in IBD and EAE samples. These shared IRGs, which encode inflammatory cytokines, exhibit high expression levels in inflammatory macrophages in IBD and may play a significant role in the inflammatory cytokine storm in MS patients. Two potential biomarkers, SOCS3 and FPR2, were screened out with great diagnostic value for MS and IBD.

TLS and immune cell profiling: immunomodulatory effects of immunochemotherapy on tumor microenvironment in resectable stage III NSCLC.

The use of programmed death-1 (PD-1) inhibitors in the neoadjuvant setting for patients with resectable stage III NSCLC has revolutionized this field in recent years. However, there is still 40%-60% of patients do not benefit from this approach. The complex interactions between immune cell subtypes and tertiary lymphoid structures (TLSs) within the tumor microenvironment (TME) may influence prognosis and the response to immunochemotherapy. This study aims to assess the relationship between immune cells subtypes and TLSs to better understand their impact on immunotherapy response. This study initially compared the tertiary lymphoid structures (TLSs) density among patients who underwent immunochemotherapy, chemotherapy and upfront surgery using 123 tumor samples from stage-matched patients. Multiplex immunohistochemistry (mIHC) was employed to analyze the spatial distribution of PD-L1+CD11c+ cells and PD1+CD8+ T cells within TLSs. Cytometry by time-of-flight (CyTOF) was used to assess immune cell dynamics in paired biopsy and resection specimens from six patients who underwent immunochemotherapy. Key immune cells were validated in newly collected samples using flow cytometry, mIHC, and in vitro CAR-T cells model. Patients who underwent neoadjuvant chemotherapy or immunochemotherapy exhibited increased TLSs compared to those who opted for upfront surgery. The TLS area-to-tumor area ratio distinguished pCR+MPR and NR patients in the immunochemotherapy group. Spatial analysis revealed variations in the distance between PD-L1+CD11c+ cells and PD1+CD8+ T cells within TLSs in the immunochemotherapy group. CyTOF analysis revealed an increase in the frequency of key immune cells (CCR7+CD127+CD69+CD4+ and CD38+CD8+ cells) following combined therapy. Treatment responders exhibited an increase in CCR7+CD4+ Tcells,whereas CD38+CD8+ T cells were associated with compromised treatment effectiveness. Immunochemotherapy and chemotherapy increase TLSs and granzyme B+ CD8+ T cells in tumors. The TLS area-to-tumor ratio distinguishes responders from non-responders, with PD-L1+ dendritic cells near CD8+PD-1+ T cells linked to efficacy, suggesting that PD-1 inhibitors disrupt harmful interactions. Post-immunochemotherapy, CD8+ T cells increase, but CD38+CD8+ T cells show reduced functionality. These findings highlight the complex immune dynamics and their implications for NSCLC treatment.

STING-activating photo-vaccination with glutamine metabolism reprogramming and cascade mitochondrial dysfunction for robust immune landscape remodeling

Autologous tumor cells hold great promise as personalized therapeutic vaccines. Nevertheless, the metabolic competition between tumor cells and the functional immune cells limits patient responses to autologous vaccine via fostering immunosuppressive microenvironment and facilitating immune escape. Herein, the STING-activating Photo-vaccination Inducer (BMA) is developed which can shift in immunological state from “cold” to “hot” by tumor metabolic reprogramming and the release of autologous tumor vaccines. BMA can form into peroxidase mimics in situ upon external energy stimulation,enabling continuous production of reactive oxygen species (ROS) through photodynamic effects and photosensitizer recycling. Tumor metabolic reprogramming with continually ROS generation facilitates maturation of dendritic cells (DCs) and reeducation of tumor immune landscapedue tospatiotemporallycascading mitochondrial and nucleus dysfunction, leading to in situ nucleic acid vaccine release and stimulator of the interferon genes (STING) pathway activation. As anticipated, results from Cytometry by Time-Of-Flight (CyTOF) results indicate that the tumor landscape has been altered, effectively eradicating primary tumors and inducing beneficial “Abscopal Effects” that counteract checkpoint blockade (ICB) resistance. Our work for the first time leverages a universal and novel strategy of free radical therapy and personalized therapeutic vaccine for tumor immunoscape re-education thereby enhancing αPD-L1 blockade, presenting a rational way to surmount immunotherapy barriers to available clinical treatments.

TMIC-80. HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC TUMOR MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Gliobastoma (GBM) is a highly aggressive solid tumor of the brain, characterized by a hypoxic tumor microevironment (TME) leading to histopathological features of necrosis, microvascular proliferation and deregulated hypervascularization. The heterogeneous nature of GBM results in a gradient of intra-tumoral hypoxia that causes molecular changes to specific cell populations within the bulk of the tumor. Our study is the first clinical study to utilize the exogenous oxygen-independent marker pimonidazole (PIMO) to identify the hypoxic TME using high-dimensional spatial and single-cell proteomics of specific cell populations within GBM. A cohort of 35 patients with primary GBM, recurrent GBM or IDH-mutant glioma were administered PIMO prior to surgical resection of tumor tissue for downstream bulk proteomic analysis of bulk tissue by serial immunohistochemistry and imaging mass cytometry (IMC) and single-cell analysis of dissociated tissue by Cytometry by time-of-flight (CyTOF). We utilized high-resolution imaging analyses to validate PIMO as a sensitive and stable marker for hypoxia against a panel of transiently expressed HIF-target genes and examine the inter- and intra-tumoral heterogeneity of hypoxia within each tumor subtype. A custom CyTOF marker panel was developed for further single-cell analyses of T-cell subsets and their functional states, myeloid cell subpopulations, natural killer cells, glial cells, and tumor cells in the hypoxic TME. We demonstrate the utility of PIMO to effectively study the hypoxic TME through spatial and single cell methods and further elucidate mechanisms of hypoxia-driven treatment resistance.

ACE2 Enhances Sensitivity to PD-L1 Blockade by Inhibiting Macrophage-Induced Immunosuppression and Angiogenesis.

Anti-PD-L1-based combination immunotherapy has become the first-line treatment for unresectable hepatocellular carcinoma (HCC). However, the objective response rate is lower than 40%, highlighting the need to identify mechanisms of tolerance to immune checkpoint inhibitors and accurate biomarkers of response. Here, we employed next-generation sequencing to analyze HCC samples from 10 patients receiving anti-PD-L1 therapy. Activation of the renin-angiotensin system was elevated in nonresponders compared with responders, and ACE2 expression was significantly downregulated in nonresponders. ACE2 deficiency promoted HCC development and anti-PD-L1 resistance, whereas ACE2 overexpression inhibited HCC progression in immune competent mice. Mass cytometry by time of flight (CyTOF) revealed that ACE2 deficient murine orthotopic tumor tissues featured elevated M2-like tumor-associated macrophages (TAMs), displayed a CCR5+PD-L1+ immunosuppressive phenotype, and exhibited high VEGFα expression. ACE2 downregulated tumor intrinsic CCL5 expression by suppressing NF-κB signaling through the ACE2/angiotensin-(1-7)/Mas receptor axis. The lower CCL5 levels led to reduced activation of the JAK-STAT3 pathway and suppressed PD-L1 and VEGFα expression in macrophages, blocking macrophage infiltration and M2-like polarization. Pharmacological targeting of CCR5 using maraviroc enhanced the tumor suppressive effect of anti-PD-L1 therapy. Together, these findings suggest that activation of the ACE2 axis overcomes the immunosuppressive microenvironment of HCC and may serve as an immunotherapeutic target and predictive biomarker of response to PD-L1 blockade.

Myeloid derived suppressor cells mediate hepatocyte proliferation and immune suppression during liver regeneration following resection.

Liver regeneration following resection is a complex process relying on coordinated pathways and cell types in the remnant organ. Myeloid-Derived Suppressor Cells (MDSCs) have a role in liver regeneration-related angiogenesis but other roles they may play in this process remain to be elucidated. In this study, we sought to examine the effect of G-MDSCs on hepatocytes proliferation and immune modulation during liver regeneration. Global gene expression profiling of regenerating hepatocytes in mice with CD11b+Ly6G+ MDSCs (G-MDSCs) depletion revealed disrupted transcriptional progression from day one to day two after major liver resection. Key genes and pathways related to hepatocyte proliferation and immune response were differentially expressed upon MDSC depletion. Hepatocytes cellularity increased when co-cultured with G-MDSCs, or treated with amphiregulin, which G-MDSCs upregulate during regeneration. Cytometry by time-of-flight (CyTOF) analysis of the intra-liver immune milieu upon MDSC depletion during regeneration demonstrated increased natural killer cell proportions, alongside changes in other immune cell populations. Taken together, these results provide evidence that MDSCs contribute to early liver regeneration by promoting hepatocyte proliferation and modulating the intra-liver immune response, and illuminate the multifaceted role of MDSCs in liver regeneration.

ImmCellTyper facilitates systematic mass cytometry data analysis for deep immune profiling.

Mass cytometry is a cutting-edge high-dimensional technology for profiling marker expression at the single-cell level, advancing clinical research in immune monitoring. Nevertheless, the vast data generated by cytometry by time-of-flight (CyTOF) poses a significant analytical challenge. To address this, we describe ImmCellTyper (https://github.com/JingAnyaSun/ImmCellTyper), a novel toolkit for CyTOF data analysis. This framework incorporates BinaryClust, an in-house developed semi-supervised clustering tool that automatically identifies main cell types. BinaryClust outperforms existing clustering tools in accuracy and speed, as shown in benchmarks with two datasets of approximately 4 million cells, matching the precision of manual gating by human experts. Furthermore, ImmCellTyper offers various visualisation and analytical tools, spanning from quality control to differential analysis, tailored to users' specific needs for a comprehensive CyTOF data analysis solution. The workflow includes five key steps: (1) batch effect evaluation and correction, (2) data quality control and pre-processing, (3) main cell lineage characterisation and quantification, (4) in-depth investigation of specific cell types; and (5) differential analysis of cell abundance and functional marker expression across study groups. Overall, ImmCellTyper combines expert biological knowledge in a semi-supervised approach to accurately deconvolute well-defined main cell lineages, while maintaining the potential of unsupervised methods to discover novel cell subsets, thus facilitating high-dimensional immune profiling.

Voluntary exercise sensitizes cancer immunotherapy via the collagen inhibition-orchestrated inflammatory tumor immune microenvironment

Physical activity reduces cancer-associated mortality through multiple mechanisms, including tumor immune microenvironment (TIME) reprogramming. However, whether and how physiological interventions promote anti-tumor immunity remain elusive. Here, we report that clinically relevant voluntary exercise promotes muscle-derived extracellular vesicle (EV)-associated miR-29a-3p for tumor extracellular matrix (ECM) inhibition in patients and mouse models, thereby permitting immune cell infiltration and immunotherapy. Mechanistically, an unbiased screening identifies EV-associated miR-29a-3p in response to leisure-time physical activity or voluntary exercise. MiR-29a-3p-containing EVs accumulate in tumors and downregulate collagen composition by targeting COL1A1. Gain- and loss-of-function experiments and cytometry by time of flight (CyTOF) demonstrate that myocyte-secreted miR-29a-3p promotes anti-tumor immunity. Combining immunotherapy with voluntary exercise or miR-29a-3p further enhances anti-tumor efficacy. Clinically, miR-29a-3p correlates with reduced ECM, increased Tcell infiltration, and response to immunotherapy. Our work reveals the predictive value of miR-29a-3p for immunotherapy, provides mechanistic insights into exercise-induced anti-cancer immunity, and highlights the potential of voluntary exercise in sensitizing immunotherapy.

Immune-related events in individuals with solid tumors on immunotherapy associate with Th17 and Th2 signatures.

BACKGROUNDImmune-related adverse events (irAEs) and their associated morbidity/mortality are a key concern for patients receiving immune checkpoint inhibitors (ICIs). Prospective evaluation of the drivers of irAEs in a diverse pan-tumor cohort is needed to identify patients at greatest risk and to develop rational treatment and interception strategies.METHODSIn an observational study, we prospectively collected blood samples and performed regular clinical evaluations for irAEs in patients receiving ICI therapy as standard of care for solid tumors. We performed in-parallel analysis of cytokines by Luminex immunoassay and circulating immune cells by cytometry by time-of-flight (CyTOF) at baseline and on treatment to investigate mechanisms of irAEs.RESULTSWe enrolled 111 patients, of whom 40.5% developed a symptomatic irAE (grade ≥ 2). Development of a grade ≥ 2 irAE was positively associated with the use of combination ICI and a history of an autoimmune disorder. Early changes in T helper 17 (Th17) (IL-6, IL-17f), type 2 (IL-5, IL-13, IL-25), and type 1 (TNF-α) cytokine signatures and congruent on-treatment expansions of Th17 and Th2 effector memory (Th2EM) T cell populations in peripheral blood were positively associated with the development of grade ≥2 irAEs. IL-6 levels were also associated with inferior cancer-specific survival and overall survival.CONCLUSIONSIn a diverse, prospective pan-tumor cohort, Th17 and Th2 skewing during early ICI treatment was associated with the development of clinically relevant irAEs but not antitumor responses, providing possible targets for monitoring and therapeutic interventions.FUNDINGJohns Hopkins Bloomberg-Kimmel Institute for Cancer Immunotherapy, the NCI SPORE in Gastrointestinal Cancers (P50 CA062924), NCI grant (R50CA243627 to LD), the NIH Center Core Grant (P30 CA006973), Swim Across America (to MY), NIAMS (K23AR075872 to LC), and imCORE-Genentech grant 137515 (to Johns Hopkins Medicine on behalf of MY).

Comprehensive Characterization of Islet Remodeling in Development and in Diabetes Using Mass Cytometry.

The pancreatic islet is the functional and structural unit of the pancreatic endocrine portion. Islet remodeling occurs in both normal development and pathogenesis of type 1 (T1D) and type 2 diabetes (T2D). However, accurately quantifying changes in islet cellular makeup and hormone expressions poses significant challenges due to large intra- and inter-donor heterogeneity and the limited scalability of traditional methods such as immunostaining. The cytometry by time-of-flight (CyTOF) technology enables simultaneous quantification of more than 30 protein markers at single-cell resolution in a high-throughput fashion. Moreover, with distinct DNA and viability markers, single live cells can be explicitly selected in CyTOF. Here, leveraging the CyTOF data generated by the Human Pancreas Analysis Program, we characterized more than 12 million islet cells from 71 donors. Our data revealed continued age-related changes in islet endocrine cell compositions, but the maturity of endocrine cells is reached by 3 years of age. We also observed significant changes in beta cell numbers and key protein expressions, along with a significant increase in bihormonal cells in T1D donors. In contrast, T2D donors exhibited minimal islet remodeling events. Our data shine a light on the islet dynamics during development and diabetes pathogenesis and suggest divergent pathogenesis processes of T1D and T2D. Our comprehensive approach not only elucidates islet plasticity but also establishes a foundation for integrated CyTOF analysis in islet biology and beyond.

Sample multiplexing in CyTOF: Path to optimize single-cell proteomic profiling

Sample multiplexing significantly enhanced the depth of single-cell proteomic analysis in CyTOF (Cytometry by Time-Of-Flight). New polymer-based chelators have broadened the utility of metal isotopes, enabling improved tagging and simultaneous analysis of multiple samples. These approaches minimize batch effects, streamline experiments, conserve valuable samples, reduce costs, enhance throughput, and increase the accuracy of biological data, thereby facilitating novel discoveries.

T-cell responses in colorectal peritoneal metastases are recapitulated in a humanized immune system mouse model.

The occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC. We studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF). The human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses. In conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.

High-dimensional mapping of human CEACAM1 expression on immune cells and association with melanoma drug resistance

BackgroundHuman carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is an inhibitory cell surface protein that functions through homophilic and heterophilic ligand binding. Its expression on immune cells in human tumors is poorly understood.MethodsAn antibody that distinguishes human CEACAM1 from other highly related CEACAM family members was labeled with 159Tb and inserted into a panel of antibodies that included specificity for programmed cell death protein 1 (PD1) and PD-L1, which are targets of immunotherapy, to gain a data-driven immune cell atlas using cytometry by time-of-flight (CyTOF). A detailed inventory of CEACAM1, PD1, and PD-L1 expression on immune cells in metastatic lesions to lymph node or soft tissues and peripheral blood samples from patients with treatment-naive and -resistant melanoma as well as peripheral blood samples from healthy controls was performed.ResultsCEACAM1 is absent or at low levels on healthy circulating immune cells but is increased on immune cells in peripheral blood and tumors of melanoma patients. The majority of circulating PD1-positive NK cells, innate T cells, B cells, monocytic cells, dendritic cells, and CD4+ T cells in the peripheral circulation of treatment-resistant disease co-express CEACAM1 and are demonstrable as discrete populations. CEACAM1 is present on distinct types of cells that are unique to the tumor microenvironment and exhibit expression levels that are highest in treatment resistance; this includes tumor-infiltrating CD8+ T cells.ConclusionsTo the best of our knowledge, this work represents the first comprehensive atlas of CEACAM1 expression on immune cells in a human tumor and reveals an important correlation with treatment-resistant disease. These studies suggest that agents targeting CEACAM1 may represent appropriate partners for PD1-related pathway therapies.

CD161+CD127+CD8+ T cell subsets can predict the efficacy of anti-PD-1 immunotherapy in non-small cell lung cancer with diabetes mellitus

ABSTRACT The role of CD161+CD127+CD8+ T cells in non-small cell lung cancer (NSCLC) patients with diabetes remains unexplored. This study determined the prevalence, phenotype, and function of CD8+ T cell subsets in NSCLC with diabetes. We recruited NSCLC patients (n = 436) treated with anti-PD-1 immunotherapy as first-line treatment. The progression-free survival (PFS), overall survival (OS), T cells infiltration, and peripheral blood immunological characteristics were analyzed in NSCLC patients with or without diabetes. NSCLC patients with diabetes exhibited shorter PFS and OS (p = 0.0069 and p = 0.012, respectively) and significantly lower CD8+ T cells infiltration. Mass cytometry by time-of-flight (CyTOF) showed a higher percentage of CD161+CD127+CD8+ T cells among CD8+T cells in NSCLC with diabetes before anti-PD-1 treatment (p = 0.0071) than that in NSCLC without diabetes and this trend continued after anti-PD-1 treatment (p = 0.0393). Flow cytometry and multiple-immunofluorescence confirmed that NSCLC with diabetes had significantly higher CD161+CD127+CD8+ T cells to CD8+T cells ratios than NSCLC patients without diabetes. The RNA-sequencing analysis revealed immune-cytotoxic genes were reduced in the CD161+CD127+CD8+ T cell subset compared to CD161+CD127−CD8+ T cells in NSCLC with diabetes. CD161+CD127+CD8+ T cells exhibited more T cell-exhausted phenotypes in NSCLC with diabetes. NSCLC patients with diabetes with ≥ 6.3% CD161+CD127+CD8+ T cells to CD8+T cells ratios showed worse PFS. These findings indicate that diabetes is a risk factor for NSCLC patients who undergo anti-PD-1 immunotherapy.CD161+CD127+CD8+ T cells could be a key indicator of a poor prognosis in NSCLC with diabetes. Our findings would help in advancing anti-PD-1 therapy in NSCLC patients with diabetes.

IGATE analysis improves the interpretability of single-cell immune landscape of influenza infection.

Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single-cell immune landscape of influenza infection. Cytometry by time-of-flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high-dimensional data remains difficult. We have developed a new analytical approach, in silico gating annotating training elucidating (iGATE), based on probabilistic support vector machine classification. By rapidly and accurately "gating" tens of millions of cells in silico into user-defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared with BALB/c was associated with a more rapid accumulation of inflammatory cell types and decreased IL-10 expression. Furthermore, we demonstrate that the most prominent effect of aging is a defective T cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibited differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varied with host genotype or aging. The software is available at https://github.com/UmichWenLab/iGATE.

Abstract PO1-13-03: Tumour infiltrating double negative (CD20+CD27- IgD-) B cells in high-risk early breast cancers. Friend or Foe??

Abstract Background: B cells exhibit diverse phenotypes and function and the complex interplay between different B cell subsets in the context of chemotherapy treated breast cancers remains unclear. Here, we investigate the dynamic changes in the B cell immune landscape before and after NACT treatment across different breast cancer subtypes. Methods: Treatment naïve, mid-treatment and post-NACT breast tumour, matched lymph node, blood and serum samples were profiled for B cell subsets and cytokines by flow cytometry, cytometry by time-of-flight (CyTOF) and Luminex technologies. Ex vivo autologous tumour organoid and immune cells cocultures to functionally assess the B-cell interactions across breast cancer subtypes were performed. Multiplex spatial analysis method was used to explore the dynamics of double negative B cell cellular crosstalk within the tumour and axillary lymph nodes. The results are being associated with treatment outcomes. Expression and activity of purinergic ectoenzymes within B cell subsets were also assessed by flow cytometry and high-performance liquid chromatography. Current work focusses on how these findings may relate to immunotherapy response, a new paradigm shift in the treatment of triple negative breast cancer patients. Results: We observed a significant expansion in the CD20+CD27−IgD− cells, also termed as double-negative (DN) B cells, within the TME of treatment naïve and post-neoadjuvant residual tumours compared to the periphery. Specifically, DN1 (CD20+ CD27−IgD− CD21+CXCR5+) cells represented the majority of DN B cells within the treatment naïve TME and in circulation. However, NACT promotes an expansion of the DN2 (CD20+CD27−IgD−CD21-CXCR5-) subset as well as a relatively uncharacterised DN3 (CD19+IgD−CD27−CXCR5−CD11c−) cell population. Preliminary analyses on how these changes (especially the enrichment of DN2 cells) relate to treatment response show significant expansion of DN2 cells in the periphery of poor responders to NACT, coupled with significantly reduced infiltration of DN2 into resistant residual tumours. Among circulating B, T and NK cells, B cells showed the highest CD73 and CD39 surface expression at baseline, but their expression significantly drops following NACT especially within the DN2 B cell subset. These ecto-5’-nucleotidases are known to play a critical role in B and T cell interactions. Spatial analyses between these cell types is ongoing. Conclusion: We report for the first time an enrichment of DN cells in breast cancer tumour tissue, specifically the expansion of DN2 cells following neoadjuvant chemotherapy. Functional analyses of tumour-infiltrated B-cells suggest that mechanistically, these B-cell subgroups may contribute to immunosurveillance and point to an important role of purinergic signalling in early breast cancers. Our study highlights the requirement for further investigation into the role of DN B cells in the context of chemo/immunotherapy resistance in breast cancers. Citation Format: Esme Carpenter, Paul Buckley, Thanussuyah Alaguthurai, Rosalind Graham, Helen Kakkassery, Gheed Alhity, Farhana Hossain, Sadiyah Mukhtar, Sheeba Irshad. Tumour infiltrating double negative (CD20+CD27- IgD-) B cells in high-risk early breast cancers. Friend or Foe?? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-03.

Impact on in-depth immunophenotyping of delay to peripheral blood processing.

Peripheral blood mononuclear cell (PBMC) immunophenotyping is crucial in tracking activation, disease state, and response to therapy in human subjects. Many studies require the shipping of blood from clinical sites to a laboratory for processing to PBMC, which can lead to delays that impact sample quality. We used an extensive cytometry by time-of-flight (CyTOF) immunophenotyping panel to analyze the impacts of delays to processing and distinct storage conditions on cell composition and quality of PBMC from seven adults across a range of ages, including two with rheumatoid arthritis. Two or more days of delay to processing resulted in extensive red blood cell contamination and increased variability of cell counts. While total memory and naïve B- and T-cell populations were maintained, 4-day delays reduced the frequencies of monocytes. Variation across all immune subsets increased with delays of up to 7 days in processing. Unbiased clustering analysis to define more granular subsets confirmed changes in PBMC composition, including decreases of classical and non-classical monocytes, basophils, plasmacytoid dendritic cells, and follicular helper T cells, with each subset impacted at a distinct time of delay. Expression of activation markers and chemokine receptors changed by Day 2, with differential impacts across subsets and markers. Our data support existing recommendations to process PBMC within 36 h of collection but provide guidance on appropriate immunophenotyping experiments with longer delays.